SAMPLE COLLECTION AND STRAIN CONFIRMATION
The thirty-five GBS strains used in this study were clinical isolates of GBS all resistant to both tetracycline (MIC, ≥ 8µg/ml) and erythromycin (MIC, ≥1µg/ml) from blood of neonates presenting with symptoms of sepsis according to WHO case definition of neonatal septicaemia. The strains were confirmed using standard bacteriological and biochemical screening such as CAMP test, Bacitracin test, sugar fermentation test. All9 GBS isolates were tested for multidrug resistance using some of the conventional antibiotics from the WHO model lists of essential medicine for children [13].
PLANTS MATERIALS AND PREPARATION OF BIOFRACTIONS
The two medicinal plants, Bryophyllum pinnatum and Rauvolfia vomitoria were collected from Oye-Ekiti, Ekiti State (Latitude 7.78N and Longitude 5.32E). The plants were taxonomically identified and authenticated at the herbarium of the department of Botany, Ekiti State University with the voucher number UHAE 2019/819 and UHAE 2019/814 respectively. The voucher specimens were deposited there for further references. The plant samples were air-dried for 3 weeks and pulverized to fine powdered using industrial blender (pharma model number 001). Fifty grams of powdered samples of each plant was extracted with ethanol, methanol, n-hexane and water using a soxhlet extractor at 60oC-100oC for 4-8hours [14]. The extracts were concentrated using Rotary Evaporator (SENCO Technology Co. Ltd. Model No: W2-1005). The crude extracts were stored in sterile McCartney bottles and refrigerated at 4oC. The stock solutions were then diluted with 5% DMSO to get three varied concentrations (25mg/ml, 50mg/ml and 100mg/ml). The extracts were tested for sterility using Millipore filtration. Column chromatography of the different concentrations of stock solution of different medicinal plants was done using different solvents (ethanol, methanol, n-hexane and water) and the separated bioactive fractions of different polarities were collected. Microbe-free proof of bioactive fractions was done according to [15].
QUALITATIVE AND QUANTITATIVE PHYTOCHEMICAL SCREENING
Phytochemical screening was carried out on the ethanol crude extracts of the selected medicinal plants using standard methods as described by [16, 17]. Phytochemical screening reveals the presence of saponin, cyanide, tannin, phytate, oxalate and alkaloids. Total alkaloid contents of the ethanol extracts of both plants were determined using [18] with slight modifications. The total oxalate and phytate content were determined by adopting the method described by [19]. Total tannin content was determined using the Folin-Dennis specrophotometric method as described by [20]. The determination of the total cyanide content and saponin content was carried out using the alkaline titration method of Association of Official Analytical chemists [21] and the method described by [22].
ANTIBIOTICS SUSCEPTIBILIY PATTERN
Kirby - Bauer disc diffusion method was used to screen for antibiotic susceptibility test using the method described by [23] with slight modification. All tests were performed in triplicates and the antibacterial activity was expressed as mean diameter zones (mm) produced by the antibiotics.
ANTIBACTERIAL SUSCEPTIBLITY TESTING ON THE BIOACTIVE FRACTIONS
Antibacterial activity was screened using the disc diffusion assay [24]. The GBS strains was first adjusted to 0.5 McFarland standards and then inoculated uniformly onto the surface of sterile Mueller Hinton agar using sterile swab sticks. Sterile filter paper disc (whatman no 1, England, 6mm diameter) were impregnated/prewetted with 10µl of the biofraction prepared at a concentration of 25mg/ml, 50mg/ml and 100mg/ml and then placed on the inoculated plates at an appropriate distance from each other. Sterile filter paper discs impregnated with DMSO was used as the negative control and amoxicillin 50mg/ml was used as positive control. All plates were left for 30min at room temperature to allow the absorption of the fraction and then incubated at 37oC for 24 h. After the incubation period, the mean diameter of inhibition halo was measured in millimeters [25]. Depending on the size of the zone and adopted assessment criteria according to [26] microorganisms were defined as sensitive or resistant. The Minimum Inhibitory Concentration (MIC) was carried out using a 5-fold serial dilution of the bioactive fraction stock with the highest activity (100mg/ml) bringing the final concentration to 100mg/ml,50mg/ml, 25mg/ml, 12.5mg/ml and 6.25mg/ml. The lowest concentration without visible growth was reported as MIC. The MBC was tested by pour plating the tube content without visible growth onto sterile Mueller Hinton agar plates and then incubated at 37oC for 48 h. MBCs were recorded as the lowest concentration of extracts that did not yield any growth or yielded less than ten pure colonies after incubation period. The MIC and MBC values were determined in duplicate.
MOLECULAR IDENTIFICATION OF TET(O) AND ERM(B) GENES
A duplex polymerase chain reaction (PCR) technique of [4] was used to identify the erythromycin and tetracycline resistant genes on ten representative strains using primers specific for Streptococcus agalactiae tet(O) and erm(B) (table 1), at Molecular and Biotechnology Laboratory of Federal University of Technology (FUTA), Akure, Nigeria. Ribonucleic acid (RNA) was isolated manually from the bacteria cells in an agar using total RNA purification kit (NorgenBiotek Corp, Thoroid , Canada). The lysis of the bacteria cells was performed using lysozyme-containing TE buffer [3 mg/ml lysozyme in (50 mMTris base, 10 mM EDTA buffer; pH 7.4). The concentration and purity of isolated RNA was determined at A260 nm and A260/A280 nm absorption.
Complementary deoxyribonucleic Acid (cDNA) Synthesis was done using FIRE Script RT cDNA Synthesis kit from Solis BioDyne (Tartus, Estonia) in a 3-step reaction condition: 25oC for 10 min, 42oC for 30 min and 85oC for 5 min. The synthesized cDNA was stored at -20oC for further downstream application. Primers specific for Streptococcus agalactiae etet(O) and erm(B) resistant genes were used. Strains were screened for the detection of resistance genes tet(O) and erm(B) using Polymerase Chain Reaction (PCR). The selection of the strains was based on the antibiotic susceptibility profiles. Polymerase Chain Reaction (PCR) for the amplification of gene of interest was performed using 5x FIREPol® Master Mix kit from Solis BioDyne (Tartus, Estonia) in a thermocycler (Primer 96 PCR- system MWG genomic Technology) according to manufacturer’s protocol.
The PCR conditions used are as follows; 95oC for 5 minutes (initial denaturation), 40 cycles of 95oC for 30 seconds (denaturation), 56oC for 30 seconds (annealing), 72oC for 1 minute (extension) and 1 cycle of 72oC for 10 minutes (final extension) followed by cooling and holding at 4oC respectively. To check whether the PCR generated the anticipated DNA fragment (amplicon), agarose gel electrophoresis was employed for size separation for the PCR products.
Table 1: Primers for PCR assay
S/N
|
Gene
|
Sequence
|
Start
|
Length
|
Tm
|
GC%
|
Amplicon Size
|
Genebank Accession Number
|
1
|
Str-aga-ermB
|
Forward: CCGTGCGTCTGACATCTATC
Reverse: ATCTGGAACATCTGTGGTATGG
|
293
494
|
20
22
|
61.904
61.813
|
55
45.455
|
202
|
EF422365.1
|
2
|
Str-aga-tetO
|
Forward: GAACAGTGGGATGCGGTAAT
Reverse: CCTTCAGGCCGTTGATGAATA
|
677
897
|
20
21
|
62.229
62.245
|
50
47.619
|
221
|
EF472563.1
|
Statistical Analysis
Results calculated from replicate data were expressed as means±standard error (SEM). Graphing was performed using Graph Pad Prism (ver.5.0a).